MOLECULAR AND CELLULAR BIOLOGY, May 2010, p. 2449–2459
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 30, No. 10
Human AlkB Homolog ABH8 Is a tRNA Methyltransferase Required
for Wobble Uridine Modification and DNA Damage Survival?
Dragony Fu,1,2,4,5Jennifer A. N. Brophy,1,4Clement T. Y. Chan,1,3,4Kyle A. Atmore,1,4
Ulrike Begley,6Richard S. Paules,7Peter C. Dedon,1,3,4
Thomas J. Begley,6and Leona D. Samson1,2,4,5*
Departments of Biological Engineering,1Biology,2and Chemistry,3Center for Environmental Health Sciences,4and Koch Institute for
Cancer Research,5Massachusetts Institute of Technology, Cambridge, Massachusetts 02138; Department of Biomedical Sciences,
GeNYsis Center for Excellence in Cancer Genomics, University at Albany, State University of New York, Rensselaer, New York 121446;
and National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 277097
Received 14 December 2009/Returned for modification 15 January 2010/Accepted 8 March 2010
tRNA nucleosides are extensively modified to ensure their proper function in translation. However, many of
the enzymes responsible for tRNA modifications in mammals await identification. Here, we show that human
AlkB homolog 8 (ABH8) catalyzes tRNA methylation to generate 5-methylcarboxymethyl uridine (mcm5U) at
the wobble position of certain tRNAs, a critical anticodon loop modification linked to DNA damage survival.
We find that ABH8 interacts specifically with tRNAs containing mcm5U and that purified ABH8 complexes
methylate RNA in vitro. Significantly, ABH8 depletion in human cells reduces endogenous levels of mcm5U in
RNA and increases cellular sensitivity to DNA-damaging agents. Moreover, DNA-damaging agents induce
ABH8 expression in an ATM-dependent manner. These results expand the role of mammalian AlkB proteins
beyond that of direct DNA repair and support a regulatory mechanism in the DNA damage response pathway
involving modulation of tRNA modification.
The posttranscriptional modification of rRNA, tRNA, and
other RNAs is critical for proper RNA folding, stability, and
function (22). These modifications range from simple methyl-
ation of the sugar or base to complex modifications involving
multiple enzymatic steps (10, 21). In tRNA alone, more than
100 different nucleoside modifications that are conserved in all
kingdoms of life have been identified. A wide variety of mod-
ified nucleosides are located in the tRNA anticodon loop,
which plays a fundamental role in decoding the genetic infor-
mation contained within mRNAs. Many of these modifications
ensure precise translation by maintaining the correct reading
frame or by directing codon specificity through stabilization of
codon-anticodon pairing (2). Recent studies uncovered a
biological role for modified wobble uridines in the regulated
translation of specific codons that are enriched in mRNAs
encoding stress and DNA damage response proteins (7).
In the yeast Saccharomyces cerevisiae, the last step in the
formation of this particular 5-methylcarboxymethyl uridine
(mcm5U) modification is catalyzed by the Trm9p methyltrans-
ferase; deletion of TRM9 diminishes the translation of specific
stress response proteins, thus conferring increased sensitivity to
DNA-damaging agents (6, 7, 26). tRNA modifications can thus
serve critical regulatory functions by modulating the transla-
tion of a subset of mRNAs (31).
Two potential human orthologs of the yeast Trm9p tRNA
methyltransferase have been identified by sequence homology,
namely, the C8ORF79 gene product and the human AlkB
homolog protein, ABH8, encoded by the ABH8 (or ALKBH8)
gene (7, 42). Notably, in addition to the methyltransferase
domain, human ABH8 contains a motif homologous to the
bacterial AlkB DNA/RNA repair enzyme. Based upon phy-
logenetic analysis, AlkB belongs to a conserved family of
nonheme, iron-dependent dioxygenases (3). Similar to other
AlkB homologs, ABH8 contains a dioxygenase catalytic core
domain encompassing cofactor-binding sites for iron and
2-oxoglutarate. However, ABH8 differs significantly from all
other mammalian AlkB homologs due to the fusion of an
RNA recognition motif (RRM) and an S-adenosyl-L-methi-
onine (SAM)-dependent methyltransferase (MT) motif to
the amino and carboxy termini of the AlkB oxygenase motif,
respectively (see Fig. 1A).
adenine and 3-methylcytosine and the exocyclic base lesions
ethenoadenine and ethenocytosine to normal adenine and cy-
tosine in DNA or RNA through a distinct oxidative demeth-
ylase reaction (12, 14, 45). Bacteria deficient in AlkB are ex-
tremely sensitive to alkylating agent toxicity, underscoring the
essential role of AlkB in repairing alkylation damage in vivo
(27). Based upon protein fold similarity, the human genome
encodes several proteins with AlkB dioxygenase motifs, includ-
ing eight AlkB homologs (ABH1 through ABH8) and the
recently discovered FTO (fat mass- and obesity-associated)
gene product (see Fig. 1A) (3, 19, 28, 41). Mouse gene knock-
out models of Abh2 and Abh3 showing that ABH2 is the major
repair enzyme for the in vivo reversal of 1-methyladenine (1-
meA) and 3-methylcytosine (3-meC) in genomic DNA have
been generated (39). While these studies define a biological
role for ABH2, the cellular functions and substrates for the
other mammalian AlkB homologs remain elusive.
Here, we find that human ABH8 catalyzes methylation of
tRNA to form mcm5U, a key posttranscriptional modification
* Corresponding author. Mailing address: 77 Massachusetts Ave-
nue, 56-230, Cambridge, MA 02139. Phone: (617) 258-7813. Fax: (617)
253-8099. E-mail: firstname.lastname@example.org.
?Published ahead of print on 22 March 2010.
at the wobble position of the anticodon loop. Moreover, ABH8
is required for maintaining steady-state levels of this methyl
modification in vivo, and importantly, loss of ABH8 along with
this modification increases the sensitivity of cells exposed to
DNA-damaging agents. These studies reveal a crucial role for
ABH8 in the DNA damage survival pathway through a distinct
mechanism involving the regulation of tRNA modification.
MATERIALS AND METHODS
Cell culture and stable RNAi. 293T human embryonic kidney, HeLa S3 cer-
vical carcinoma, and derivative stable cell lines were cultured in Dulbecco’s
minimal essential media (Invitrogen) containing 10% fetal bovine serum (FBS)
and 2 mM L-glutamine. For stable RNA interference (RNAi), lentiviral RNAi
constructs (Open Biosystems) expressing a nonsilencing short hairpin RNA
(shRNA) (RHS4346), one of two shRNAs targeting the ABH8 transcript
(RHS4430-99614583 and RHS4430-99616735), or a shRNA targeting the human
URM1 transcript were cotransfected with packaging plasmids (psPAX2 and
pMD2.G) into 293T cells for lentivirus production. The 293T cell line was
subsequently infected with lentivirus in the presence of Polybrene, followed by
stable clone selection using puromycin.
For colony-forming analysis, cells were seeded into 6-well plates in duplicate,
allowed to attach for 24 h, and exposed to serum-free media alone or media
containing methyl methanesulfonate (MMS) for 1 h, followed by replacement
with complete media containing serum. Surviving colonies were scored 6 to 10
days later by methylene blue staining. For short-term viability assays, cells were
exposed to either MMS (Sigma) for 1-hour in serum-free media or bleomycin
(EMD Biosciences) for 24 h in serum-containing media before analysis. Viability
in the presence of MMS was determined using a Coulter Counter coupled with
trypan blue staining (total number of viable treated cells/total number of viable
untreated cells), and viability in the presence of bleomycin was determined with
the WST-1 cell proliferation reagent (Roche) according to the manufacturer’s
Mammalian expression constructs. The coding region for the full-length iso-
form of human ABH8 was amplified by reverse transcription-PCR (RT-PCR)
(Easy-A; Stratagene) from human testis RNA (100179-748; VWR) and cloned
into pcDNA3.1 (Invitrogen) for expression as N-terminal green fluorescent pro-
tein (GFP) or triple FLAG tag fusion proteins. The splice isoforms for ABH8
correspond to the following accession numbers: RRM, NM_138775.1; RRM-
AlkB, EAW67088; MT domain alone, BX649085. The AlkB-MT variant was
PCR amplified from the full-length ABH8 construct to create a cDNA encoding
amino acids residues 126 to 664 of ABH8.
For stable expression of epitope-tagged ABH8, the FLAG-tagged-ABH8 cod-
ing region was cloned into the pBABEpuro or pBABEhygro retroviral vector for
viral packaging using Phoenix 293T cells or stable transfection (33). HeLa S3
cells were infected with retrovirus and selected in bulk with puromycin for 2 days.
The 293T ABH8-shRNA1 cell line was transfected with the pBABEhygro
FLAG-ABH8 construct and selected with hygromycin. Stable clones were ex-
panded and analyzed for expression.
Affinity purification of ABH8 from human cells. Transient transfection and
cellular extract production were performed as previously described (17). Whole-
cell extract from transiently transfected cells or stable cell lines (1 mg of total
protein) was rotated with 10 ?l of FLAG M2 antibody resin (Sigma) for 2 h at
4°C in lysis buffer (20 mM HEPES at pH 7.9, 2 mM MgCl2, 0.2 mM EGTA, 10%
glycerol, 1 mM dithiothreitol [DTT], 0.1 mM phenylmethylsulfonyl fluoride
[PMSF], 0.1% NP-40) with 150 mM NaCl. Resin was washed extensively using
the same buffer. Bound samples were eluted with two sequential volumes of wash
buffer containing 100 ?g/ml of 3?FLAG peptide (Sigma).
Protein and RNA analyses. Cellular extracts and purified protein samples were
fractionated on 4 to 12% Bis-Tris polyacrylamide gels (Invitrogen), followed by
silver staining (SilverQuest kit; Invitrogen) or transfer to a nitrocellulose mem-
brane for immunoblotting. Antibodies were against the following proteins:
FLAG (F1804; Sigma), ABH8 (AV41106; Sigma), T-complex protein 1 subunit
alpha (TCP-?) (sc-13869; Santa Cruz), TCP-? (sc-58864; Santa Cruz), TCP-?
(sc-13891; Santa Cruz), human Trm112 (also known as HSPC152; ab57156;
Abcam), Hsp60 (sc-1052; Santa Cruz), URM1 (15285-1-AP; Protein Tech
Group), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (AV00191; Gen-
Script). Primary antibodies were detected using IRDye-conjugated secondary
antibodies (Rockland). Immunoblots were scanned using direct infrared fluores-
cence via the Odyssey system (Li-Cor Biosciences).
RNA from input and purified samples was extracted using TRIzol (Invitrogen)
and fractionated on 7 M urea–0.5? Tris-borate-EDTA (TBE)–15 to 20% dena-
turing polyacrylamide gels, followed by Sybr gold nucleic acid staining (Invitro-
gen). RNA was visualized by 300 nM UV transillumination or a 450-nm light-
emitting diode (LED) on a Storm 865 gel imaging system (GE Healthcare) and
subsequently transferred to a Hybond N? membrane (GE Healthsciences) for
probe hybridization with 5?-radiolabeled oligonucleotides (Table 1).
Protein mass spectrometry. Protein identification was performed by the MIT
Center for Cancer Research Biopolymers Laboratory (http://web.mit.edu/ki
/facilities/biopolymers/). Briefly, protein samples were reduced, alkylated, and
digested in solution with trypsin, followed by purification and desalting on an
TABLE 1. Oligonucleotide sequences
aBoldface indicates degenerate bases.
2450 FU ET AL.MOL. CELL. BIOL.
analytical C18column tip. Peptide samples were analyzed by chromatography on
an Agilent model 1100 Nanoflow high-pressure liquid chromatography (HPLC)
system coupled with electrospray ionization on a Thermo Electron model LTQ
ion trap mass spectrometer. Protein identification through tandem mass spec-
trum correlation was performed using SEQUEST (30). Spectra had to match full
tryptic peptides of at least 7 amino acids, have a normalized difference in
cross-correlation scores (?Cn) of at least 0.1, and have minimum cross-correla-
tion scores (Xcorr) of 1.8 for singly charged, 2.5 for doubly charged, and 3.5 for
triply charged spectra with at least 50% ion coverage.
Methyltransferase and demethylase activity assays. The methyltransferase
assay was carried out essentially as described previously (16). Purified samples
were incubated in a total volume of 30 ?l containing 1? HpaII methyltransferase
buffer (New England Biolabs), 0.4 mM S-adenosyl-L-methionine14C labeled at
50 mCi/mmol (Perkin Elmer), 10 ?l of purified enzyme, and 400 to 500 ng of
either small RNAs purified from the 293T control shRNA or an ABH8-shRNA1
stable cell line using the PureLink microRNA (miRNA) isolation kit (Invitro-
gen), single- or double-stranded DNA consisting of a 49-mer oligonucleotide
(Table 1), or bovine serum albumin (New England Biolabs). The reaction mix-
tures were incubated at 25°C for 2 h, followed by the addition of diethyl pyro-
carbonate (DEPC)-H2O to 20 ?l and passage through a Quick Spin RNA
column (Roche) for RNA, a Quick Spin oligonucleotide column for DNA, or a
Microcon YM-30 centrifugal filter unit for protein. The eluates were added to 10
ml of scintillation fluid for scintillation counting.
Restriction enzyme-mediated oligonucleotide demethylase assays were per-
formed essentially as described previously (39). Radiolabeled DNA oligonucle-
otides (150 fmol) containing 1-meA, 3-meC, or ethenoadenine lesions (Euro-
gentec; sequences listed in Table 1) were incubated at 25°C for 1 h in a total
volume of 50 ?l containing 50 mM Tris, pH 8.0, 2 mM ascorbic acid, 0.5 mM
?-ketoglutarate, 40 ?M FeSO4, and 10 ?l of purified enzyme (?50 ng protein)
or buffer, followed by heat inactivation at 70°C for 15 min. An aliquot of each
reaction mixture was digested with DpnII (New England Biolabs) for 1 h at 37°C,
followed by electrophoresis on 7 M urea–18% denaturing polyacrylamide gels for
1 h at 500 V. Gels were visualized using a Cyclone phosphorimager (Perkin
ABH8 expression analysis. For ABH8 gene expression analysis, NHF1-hTert-
lenti-LACZ (control line) and NHF1-hTert-lenti-ATM5 (ATM knockdown line)
cells were constructed by methods similar to those in reference 4. Cells (4.5 ? 104
cells/well) were plated in 6-well culture dishes. Twenty-four hours after plat-
ing cells were treated with 100 ?M bleomycin (Research Products International,
Mt. Prospect, IL) for 90 min, and the drug was removed and replaced with fresh
growth medium. RNA was isolated at several time points using TRIzol reagent
(Invitrogen) and subsequently purified by ethanol precipitation. Quantitative
TaqMan PCR analysis was carried out with the ABI Prism 7900HT sequence
detection system (Applied Biosystems, Foster City, CA) using the TaqMan
one-step RT-PCR master mix reagent kit (Applied Biosystems). Assays were
performed in a reaction volume of 20 ?l, and reaction mixtures contained 500 ng
purified RNA, 1? master mix without uracil–N-glycosylase (UNG), 1? Multi-
Scribe and RNase inhibitor mix, and 1? probes and primer sets specific to
Hs01098105_m1 (hABH8) or 1? human GAPDH. Thermal cycler parameters
were as follows: incubation at 48°C for 30 min (reverse transcription step),
denaturation at 95°C for 10 min, and then 40 cycles of the amplification step
(denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min). All
amplification reactions were performed in triplicate, and the relative quantifica-
tion of ABH8 mRNA expression was determined after normalization with the
endogenous control, GAPDH. Data processing and statistical analysis were per-
formed using the ABI Prism SDS software, version 2.1 (Applied Biosystems).
Quantification of 5-carboxymethyl uridine (cm5U) and mcm5U. RNA samples
were isolated from 5 ? 106human cells using the PureLink miRNA isolation kit
(Invitrogen), with the deaminase inhibitors tetrahydrouridine (50 ?g/ml) and
coformycin (5 ?g/ml) and the antioxidants desferroxamine (0.1 mM) and butyl-
ated hydroxytoluene (0.1 mM) added to each sample to prevent postprocessing
modification or degradation. Following quantification of tRNA (Agilent; 2100
Bioanalyzer), 1 pmol of [15N]5dA was added per ?g of tRNA as an internal
standard. RNA samples were lyophilized and redissolved at a final concentration
of 120 ?g of tRNA/ml in digestion buffer (30 mM sodium acetate, 2 mM zinc
chloride, pH 6.8) with coformycin, tetrahydrouridine, desferroxamine, and bu-
tylated hydroxytoluene as noted earlier. RNA was hydrolyzed to nucleoside form
by addition of nuclease P1 (1 unit) and RNase A (5 units) and incubation for 3 h
at 37°C, followed by addition of 50 ?l sodium acetate buffer (30 mM, pH 7.8),
alkaline phosphatase (10 units), and phosphodiesterase I (0.5 unit) and overnight
incubation at 37°C. Proteins were removed from the mixtures by ultrafiltration
The chromatographic and mass spectrometric behavior of cm5U and mcm5U
was initially established with tRNA from S. cerevisiae using HPLC-coupled,
high-resolution quadrupole time-of-flight mass spectrometry (LC-QTOF; Agi-
lent; 6510) operated in positive ion mode, on the basis of calculated m/z values
of 303.0823 and 317.0979, respectively. Following hydrolysis and deproteination,
the mixture of nucleosides was resolved by reversed-phase HPLC (Thermo
Scientific; Hypersil Gold aQ, 150 by 2.1 mm, 3-?m particle size) and eluted at 0.3
ml/min and 36°C with solvent A (0.2% acetic acid in acetonitrile) and a gradient
of solvent B (0.2% acetic acid in water) as follows: 0 to 18 min, 1 to 2%; 18 to
23 min, 2%; 23 to 28 min, 2 to 7%; 28 to 30 min, 7%; 30 to 31 min, 7 to 100%;
31 to 41 min, 100%. The eluting nucleosides, cm5U and mcm5U, were identified
by scanning the HPLC eluate from m/z 100 to 1,000 for deglycosylation products
derived from collision-induced dissociation (CID). Species with m/z values of
303.0823 and 317.0979 were observed with elution times of 4.6 and 12.8 min,
respectively. The pattern of fragments produced by CID of each species revealed
deglycosylation products consistent with the structures of cm5U and mcm5U (see
Fig. 5B) and consistent with published observations (26). Analysis of yeast de-
ficient in trm9 revealed a nearly complete loss of mcm5U.
The relative quantities of cm5U and mcm5U in tRNA from the various cell
preparations were determined by HPLC-coupled tandem quadrupole mass spec-
trometry (LC-MS/MS; Agilent; 6410) using the following HPLC retention times
and molecular transitions: cm5U, 4.6 min, m/z 303 to 171; mcm5U, 12.8 min, m/z
317 to 185, and [15N]5dA, 10.1 min, m/z 257 to 141. The mass spectrometer was
operated with an electrospray ionization source operated in positive ion mode
and the following parameters: gas temperature, 350°C; gas flow, 10 liter/min;
nebulizer pressure, 20 lb/in2; capillary voltage, 3,500 V; unit resolution. The
signal intensity for each species was normalized by the signal for the [15N]5dA
Affinity purification and proteomic analysis of ABH8 com-
plexes. Human ABH8 protein is the largest identified member
of the mammalian AlkB homologs due to the fusion of an
RNA recognition motif (RRM) and an S-adenosyl-L-methio-
nine (SAM)-dependent methyltransferase (MT) motif to the
amino and carboxy termini of the AlkB oxygenase motif, re-
spectively (Fig. 1A). Among the identified AlkB homologs,
ABH8 is the only member containing these additional do-
mains. While the RRM motif represents a putative nucleic acid
binding fold found in many RNA-associated proteins, the
ABH8 MT domain is most closely related to the Trm9p tRNA
methyltransferase found in yeast (Fig. 1B; data not shown).
Phylogenetic analysis reveals ABH8 orthologs in higher eu-
karyotes, including all sequenced animal species, as well as a
paralogous protein encoded by the C8ORF79 gene (Fig. 1B).
Intriguingly, database searches have identified several ABH8
transcripts encoding different ABH8 variants encompassing
various combinations of the individual domains (Fig. 2A). The
modular domain format of the ABH8 coding exons suggests
that alternative splicing could produce functional proteins with
Green fluorescent protein tagging and expression revealed
that the largest ABH8 variant displays a primarily cytoplasmic
localization, with considerably less in the nucleus in human
cells (Fig. 1C). Analysis of the remaining ABH8 variants re-
veals a similar pattern of cytoplasmic localization (D. Fu and L.
Samson, unpublished observations). We note that these fluo-
rescence results reveal only the steady-state localization since
ABH8 could shuttle through or translocate to the nucleus
under certain conditions.
To identify ABH8-interacting factors that would link ABH8
to a particular cellular process, we used affinity purification to
isolate ABH8 complexes from human cells. Mammalian ex-
pression constructs encoding human ABH8 fused to the FLAG
epitope tag were transfected into 293T human embryonic kid-
VOL. 30, 2010 tRNA METHYLATION BY THE HUMAN AlkB HOMOLOG ABH82451
ney cells, followed by whole-cell extract preparation and affin-
ity purification on anti-FLAG antibody resin. Polyacrylamide
gel fractionation and silver staining of the purified samples
revealed several polypeptides that were detected specifically in
the purified ABH8 sample (Fig. 2B). Several ABH8-interact-
ing proteins were found in the 55- to 65-kDa range along with
a prominent low-molecular-mass protein of ?15 kDa.
After processing the eluates from parallel FLAG-ABH8 and
mock purifications as complex mixtures for peptide identifica-
tion by liquid chromatography-mass spectrometry, we com-
piled an inventory of proteins recovered specifically with
ABH8 (Table 2). As expected, numerous peptide sequences
confirmed the recovery of ABH8 itself. In addition, mass spec-
trometric analysis of the purified ABH8 samples revealed all
eight subunits of the TCP ring complex (TRiC) (Fig. 2B; Table
FIG. 1. Human ABH8 contains a tRNA methyltransferase motif
and localizes to the cytoplasm. (A) Schematic of the human AlkB
homolog (ABH) family of nonheme iron/2-oxoglutarate-dependent
dioxygenase proteins. (B) Annotated gene tree of ABH8. The maxi-
mum likelihood phylogenetic tree of ABH8 and homologous proteins
was generated via TreeBeST (tree building guided by species tree)
using the Ensembl genome database. The complete tree with bootstrap
values and distances is available on request. S. pombe, Schizosaccha-
romyces pombe; K. lactis, Kluyveryomyces lactis. (C) Subcellular local-
ization of green fluorescent protein-tagged ABH8. Human 293T em-
bryonic kidney cells were transfected with constructs expressing the
indicated proteins and visualized by fluorescence microscopy. The
nucleus was visualized by DAPI (4?,6-diamidino-2-phenylindole) stain-
ing. As a comparison, GFP alone was analyzed alongside.
FIG. 2. Purification and analysis of human ABH8 complexes.
(A) Schematic of ABH8 variants and domains. RRM, RNA recogni-
tion motif; 2OG, 2-oxoglutarate; Fe(II), iron; SAM, S-adenosyl-L-me-
thionine; AlkB, dioxygenase domain; MT, methyltransferase. (B) Pro-
tein profile of purified ABH8 complexes by silver stain analysis. Arrows
indicate proteins specifically identified in purified ABH8 samples.
(C) Confirmation and characterization of interactions between ABH8
with TRiC subunits and Trm112. (D) Purification of ABH8 complexes
from HeLa human cells.
2452FU ET AL.MOL. CELL. BIOL.
2). TRiC forms a large, cylindrical chaperone assembly that is
required for the folding of numerous cytosolic substrates (9).
However, none of the AlkB homologs or SAM-dependent
MTs was previously identified as a TRiC-associated protein.
In addition to the TRiC chaperonin, we identified a low-
molecular-mass protein of 14 kDa termed human “Trm112-
like” in the purified ABH8 samples, henceforth referred to as
Trm112 (Fig. 2B; Table 2). In the yeast Saccharomyces cerevi-
siae, the orthologous Trm112p is a zinc finger protein subunit
of three different methyltransferases, including the protein
methylase Mtq2p and the tRNA methylases Trm9p and
Trm11p (18). While Trm112p itself lacks a methyltransferase
domain and does not catalyze methylation, it is an essential
protein that is required for tRNA methylation in vivo (24, 38,
44). Like yeast Trm112, human Trm112 has been shown to
interact with the human Mtq2 ortholog (15). However, an
association between Trm112 and a human AlkB protein was
not previously described.
Interaction analysis of ABH8-associated proteins. The TRiC
chaperonin and Trm112 represent the first putative proteins
associated with the human ABH8 protein. To confirm these
proteins as bona fide components of ABH8 complexes, we
used coimmunoprecipitation followed by immunoblot analysis.
To further characterize the significance of these interactions,
we also tested the association of putative ABH8 variants with
components of TRiC and with Trm112. The known ABH8
splice variants were expressed in human cells as FLAG-tagged
fusion proteins; these were RRM alone, RRM with the dioxy-
genase motif, and the MT domain alone. In addition, we ex-
pressed the ABH8 dioxygenase motif fused to the MT domain
(Fig. 2A). Whole-cell extracts from cells expressing tagged
ABH8 proteins were subjected to affinity purification and im-
munoblot analysis. Using antibodies against TRiC compo-
nents, we detected the specific copurification of three TRiC
subunits with full-length ABH8 (Fig. 2C). Importantly, we did
not detect the abundant Hsp60 chaperone in the purified
ABH8 sample, indicating a specific interaction between ABH8
and TRiC (Fig. 2C). From the ABH8 domain purifications, we
found that TRiC could associate only with ABH8 and its vari-
ants that contain the MT domain, including the MT domain
alone (Fig. 2C). We infer that the ABH8 MT domain is nec-
essary and sufficient for association with TRiC.
Next, we probed the purified ABH8 samples for the pres-
ence of Trm112. As was found for TRiC, we detected specific
copurification of Trm112 with full-length ABH8 but not with
the control, RRM, or RRM-AlkB (Fig. 2C). However, unlike
the TRiC-ABH8 association, the ABH8 MT domain was not
sufficient for Trm112 interaction; rather, human Trm112 re-
quires both the AlkB motif and the MT domain to interact with
ABH8 since we could readily detect an association between
Trm112 and the AlkB-MT variant (Fig. 2C). Because an AlkB
homolog has not been identified in S. cerevisiae, the AlkB
motif-dependent interaction between human Trm112 and
ABH8 is distinct from the yeast Trm112p interaction with the
Trm9p tRNA methylase. Together, these results reveal TRiC
and Trm112 as potential components within an ABH8 com-
To confirm the interactions described above, we generated
HeLa human cell lines that stably express lower levels of
FLAG-tagged full-length ABH8. Using these cell lines, we
detect robust copurification of Trm112 with ABH8 in the ab-
sence of the TRiC chaperone (Fig. 2D). These results verify a
stable ABH8-Trm112 complex, which associates with TRiC
upon ABH8 overexpression. Given the stable interaction be-
TABLE 2. Proteins identified by mass spectrometry specifically in purified human ABH8 samplesa
PurificationAccession no. ProteinProbabilityb
No. of unique
Alkylated DNA repair protein AlkB homolog 8
T-complex protein 1 subunit eta
T-complex protein 1 subunit epsilon
T-complex protein 1 subunit beta
T-complex protein 1 subunit delta
Heat shock protein HSP90 alpha
T-complex protein 1 subunit theta
T-complex protein 1 subunit alpha
T-complex protein 1 subunit zeta
Chaperonin containing TCP1, subunit 3
T-complex protein 1 subunit beta
T-complex protein 1 subunit zeta
T-complex protein 1 subunit eta
Alkylated DNA repair protein AlkB homolog 8
T-complex protein 1 subunit alpha
T-complex protein 1 subunit delta
T-complex protein 1 subunit theta
T-complex protein 1 subunit epsilon
Chaperonin containing TCP1, subunit 3
aInventories of proteins were compiled from independent FLAG-ABH8 purifications compared to a mock control purification for subtraction of nonspecific protein
bProbability of the best peptide matched to a peptide spectrum of a particular protein in the human protein database.
cMW, molecular weight (in thousands).
VOL. 30, 2010tRNA METHYLATION BY THE HUMAN AlkB HOMOLOG ABH8 2453
tween ABH8 and Trm112 along with the presence of an MT
domain in human ABH8, we focused on the potential role of
ABH8 in tRNA modification.
ABH8 catalyzes RNA methylation through its methyltrans-
ferase domain. The initial identification of ABH8 as a putative
AlkB dioxygenase that also contains a SAM-dependent MT
domain suggests that ABH8 possesses multiple enzymatic ac-
tivities. However, we were unable to detect oxidative demeth-
ylation activity for any of the ABH8 variants on canonical AlkB
substrates, namely, 1-meA, 3-meC, or ethenoadenine bases
present in DNA substrates (Fig. 3). While ABH8 does not
display AlkB-like activity on these substrates, its potential tar-
gets could encompass bases in DNA or RNA that remain to be
Based upon the copurification of the Trm112 subunit with
ABH8, we next tested the purified ABH8 samples for methyl-
transferase activity using a previously described methylase as-
say (16). The purified ABH8 proteins were incubated with
DNA, RNA, or protein substrates in the presence of SAM
radiolabeled in the transferable methyl, followed by removal of
unincorporated SAM and scintillation counting of labeled
product. First, we tested RNA highly enriched for tRNAs
purified from human cells depleted of ABH8 (described be-
low) to increase the levels of potential ABH8 methyl acceptor
substrates. Control reactions with a mock purification pro-
duced very low levels of methyl transfer to RNA, DNA, or
protein. In contrast, purified full-length human ABH8 cata-
lyzed robust RNA methylase activity, with a 2.5-fold increase in
methyl transfer above background (Fig. 4; Table 3). Further-
more, the ABH8 AlkB-MT variant and the MT domain alone
catalyzed RNA methylation to levels similar to that for the
full-length ABH8 isoform. Importantly, we found that ABH8
variants lacking the MT domain yielded only background ac-
tivity levels. Using the same reaction conditions as for RNA,
we did not detect any methylase activity above background for
either single- or double-stranded DNA substrates with full-
length ABH8 (Fig. 4; Table 3). However, the ABH8 MT do-
main alone exhibited weak methylase activity on DNA, sug-
gesting that the RRM and/or AlkB motif confers substrate
specificity to the ABH8 MT domain. As a final specificity
control, we found that none of the ABH8 variants could meth-
FIG. 3. ABH8 does not demethylate canonical AlkB DNA substrates. The full-length ABH8 and domain variants were tested for oxidative
demethylase activity on DNA substrates using a restriction enzyme-mediated oligonucleotide demethylase assay (see Materials and Methods).
Purified ABH8 variants were incubated with a 49-mer DNA oligonucleotide containing 1-methyladenine (1-meA), 3-methylcytosine (3-meC), or
ethenoadenine (ethenoA) (Table 1), followed by digestion with the methylation-sensitive restriction enzyme DpnII and gel electrophoresis.
Unrepaired substrate and repaired product are indicated. The ABH2 protein, a known DNA/RNA demethylase, was included as a control.
FIG. 4. ABH8 catalyzes RNA methylation. Shown is in vitro meth-
yltransferase activity of purified ABH8 variants on the indicated sub-
strates. A mock purification sample (control) or purified ABH8 vari-
ants were incubated with purified human RNA enriched for tRNAs
(from control or ABH8-depleted cells), DNA oligonucleotides, or bo-
vine serum albumin protein followed by methyl donor removal and
measurement of the eluted samples via scintillation counting. Methyl-
transferase activity is expressed as increase relative to the control. The
average14C incorporation for each sample is listed in Table 3.
2454 FU ET AL.MOL. CELL. BIOL.
ylate bovine serum albumin protein. Significantly, the full-
length and MT domain variants of ABH8 display much greater
methyltransferase activity on RNA that is enriched for cm5U
(Fig. 4B). Combined with enzymatic assays performed with
recombinant human ABH8-Trm112 complexes purified from
bacteria (L. Songe-Møller et al., personal communications),
these results uncover a previously unidentified RNA methylase
activity for human ABH8 catalyzed by its MT domain.
ABH8 interacts with tRNAs containing known modifications
on their wobble uridines. The presence of an active RNA
methyltransferase domain in ABH8 that is homologous to
tRNA MTs, along with the copurification of Trm112, suggests
that ABH8 plays a role in the posttranscriptional modification
of tRNA. We therefore analyzed the purified ABH8 complexes
for the presence of copurifying RNAs. As expected, RNA
purified from the input cell extracts displayed a complex pat-
tern of small RNAs (Fig. 5A). In contrast, RNA purified from
the ABH8 complex ran as a single, prominent RNA species
migrating at position analogous to that for tRNAs in the total
RNA input (Fig. 5A). This RNA band is specific for ABH8
since we did not detect any RNAs in the control mock purifi-
To determine the identity of the ABH8-associated RNA mol-
ecules, they were hybridized with a panel of oligonucleotides
complementary to specific tRNAs. We classified tRNAs into four
groups based upon their canonical wobble nucleosides (U, G, C,
or inosine) and selected at least one tRNA from each group for
probing. Conspicuously, we detected two wobble uridine-con-
taining tRNAs associated with the ABH8 complex, namely,
tRNAArg(UCU)and tRNAGlu(UUC)(Fig. 5B). This interaction
was specific to these particular wobble uridine tRNAs since
two other tRNAs with wobble uridines, tRNALys(UUU)and
tRNASec(UGA), did not copurify with ABH8 (Fu and Samson,
unpublished). In contrast, none of the other tested tRNAs
containing G, C, or inosine at the wobble position copurified
with ABH8. As additional controls, we did not detect 5S
rRNA or U6 snRNA in the purified ABH8 samples. It is
important to note that yeast counterparts of tRNAGlu(UUC)
and tRNAArg(UCU)are distinguished by the presence of a
modified wobble uridine in the anticodon loop; the wobble
uridine is initially converted to cm5U, with subsequent meth-
ylation by the Trm9p tRNA methylase to form mcm5U (26).
Moreover, mcm5U and its thiolated version, mcm5s2U, has
been found in the wobble position of mammalian tRNAs as
well, including tRNAGlu(UUC), tRNAArg(UCU), tRNALys(UUU),
and tRNASec(UGA)(23, 43). In humans, the putative methylase
catalyzing this methylation event was unknown. That purified
ABH8 displays RNA methylase activity and interacts with
tRNAs subject to wobble uridine methylation suggests that
ABH8 plays a role in mcm5U formation in tRNA.
ABH8 is required for maintaining mcm5U modification. To
determine whether ABH8 is involved in tRNA modification in
vivo, we used small hairpin RNA (shRNA) to specifically de-
plete ABH8 from human cells. Two different shRNA con-
structs targeting distinct regions of the ABH8 mRNA and a
nonsilencing shRNA construct with no known human targets
were used. The retroviral shRNA constructs were integrated
into 293T human embryonic kidney cells to generate stable cell
lines expressing each shRNA. We successfully depleted ABH8
with both ABH8 shRNA constructs, as judged by immunoblot
analysis (Fig. 6A). The ABH8-depleted cell lines exhibited no
overt changes in morphology or growth rate.
FIG. 5. ABH8 interacts with a subset of tRNAs containing known
modified wobble uridine residues. (A) Denaturing gel electrophoresis
and Sybr gold stain of ABH8-associated RNAs. Arrows denote the
migration pattern of the indicated RNAs, and the asterisk represents
an ABH8-associated RNA species. (B) RNA blot hybridization anal-
ysis of ABH8-associated RNAs with the indicated oligonucleotide
probes. The presence of a modified uridine (mcm5U) in the equivalent
yeast tRNA is indicated on the right.
TABLE 3. Absolute14C incorporation values for RNA, DNA, and
protein substrates with ABH8
Substrate Form of ABH8cpma
RNA Control (buffer)
DNA (single stranded)Control (buffer)
Protein (bovine serum
DNA (double stranded) Control
aAverage counts per minute of the radiolabeled products from the indicated
VOL. 30, 2010tRNA METHYLATION BY THE HUMAN AlkB HOMOLOG ABH82455
To determine whether modulation of ABH8 levels had any
effect on mcm5U levels in cells, small RNAs highly enriched
for tRNAs were purified from the control and ABH8-depleted
cell lines and analyzed for mcm5U by mass spectrometry (Fig.
6B). Consistent with previous studies in wild-type yeast (26),
the levels of mcm5U were much greater than that of cm5U in
RNA purified from the control human cell line (Fig. 6C). In
contrast, RNA from ABH8-depleted cells displayed a dramatic
decrease in mcm5U, with a concomitant rise in the cm5U
precursor (Fig. 6C). Importantly, the increase in cm5U levels
found in the RNA of ABH8-depleted cell lines indicates that
ABH8 is required for the final methyl modification to generate
mcm5U rather than being involved in an upstream step in
modified wobble uridine formation. Taken together, our re-
sults demonstrate that human ABH8 catalyzes the last step of
mcm5U formation in vivo.
ABH8 is required for DNA damage survival. In yeast,
Trm9p-mediated wobble uridine methylation plays a critical
role in the regulated translation of stress and DNA damage
response pathway proteins (7, 26). Consequently, trm9 deletion
mutants are extremely sensitive to gamma irradiation and the
alkylating agent methyl methanesulfonate (MMS) (5–8). Based
upon our finding that ABH8 is required for wobble uridine mod-
ification in human cells, we hypothesized that mammalian ABH8
could also play an essential role in DNA damage survival.
We determined whether ABH8 influences MMS sensitivity
using colony formation and cellular viability assays. Human
cell lines depleted for ABH8 displayed sensitivity to MMS
compared to the control shRNA cell line, as measured by both
assays (Fig. 7A and B). To determine whether the sensitivity
phenotype exhibited by ABH8-depleted cells is specific to
alkylation damage, we also tested sensitivity to bleomycin, which
Bleomycin intercalates between DNA base pairs and catalyzes
free radical production to induce double-strand breaks (13). We
found that ABH8-depleted cell lines displayed significant sen-
sitivity to bleomycin, similar to their sensitivity to MMS (Fig.
7C). Thus, ABH8 increases the survival of human cells upon
exposure to at least two damaging agents, namely, MMS and
bleomycin. This sensitivity of ABH8-depleted human cells is
comparable to that observed in cells depleted of characterized
DNA damage response proteins, including the central DNA
damage kinase, ATM, and the base excision repair protein,
alkyladenine glycosylase (4, 36). Moreover, ABH8-depleted
cells display significantly more sensitivity to MMS than cells
completely devoid of ABH2, which is known to repair DNA in
To confirm the role of ABH8 in DNA damage survival, we
generated ABH8-depleted cell lines containing either an
integrated empty vector or a construct expressing a siRNA-
resistant form of the full-length ABH8 variant. Since the
ABH8 shRNA1 cell line was generated using a shRNA con-
struct targeting the 3? untranslated region (UTR) of endoge-
nous ABH8 transcripts, we utilized the ABH8 expression con-
struct described in Fig. 2 to express a transcript containing the
full-length FLAG-tagged-ABH8 open reading frame (ORF)
lacking any ABH8 3? UTR sequences. Unfortunately, we could
not detect the FLAG-ABH8 protein with the anti-ABH8 an-
tibody used in this study, most likely due to steric hindrance by
the amino-terminal FLAG tag against the antibody, which
recognizes only the first 50 amino acids of ABH8. Thus, we
confirmed stable expression of the recombinant ABH8 in the
ABH8 shRNA1 cell line using anti-FLAG antibodies (Fig.
7D). We find that expression of full-length ABH8 can partially
FIG. 6. ABH8 is required for maintaining mcm5U levels in vivo. (A) Confirmation of ABH8 depletion in human cells by immunoblot analysis.
The depletion of ABH8 is expressed as a percentage relative to the control shRNA cell line normalized to the GAPDH loading control.
(B) Identification of cm5U and mcm5U by HPLC-coupled tandem quadrupole mass spectrometry. Shown are extracted ion chromatograms of
molecular species consistent with cm5U and mcm5U nucleobases based upon theoretical m/z. (C) Detection and comparison of cm5U and mcm5U
levels in small RNAs purified from control and ABH8-depleted human cells. The levels of cm5U and mcm5U nucleosides were detected by mass
spectrometry and quantified by integration of the normalized peak intensity for each nucleoside signal.
2456 FU ET AL.MOL. CELL. BIOL.
complement the phenotype of sensitivity of ABH8-depleted
cells to either MMS or bleomycin, as demonstrated by the
increased damage resistance of ABH8-depleted cells express-
ing ABH8 compared to that of cells expressing the empty
vector alone (Fig. 7E and F). The partial rescue of DNA
damage sensitivity by full-length ABH8 could be due to the
contributions of the remaining ABH8 splice isoforms to DNA
damage survival. These results indicate that expression of at
least full-length ABH8 contributes to viability after DNA dam-
age. However, full rescue of damage sensitivity in ABH8-de-
pleted cells could require expression of multiple ABH8 vari-
The requirement for ABH8 in DNA damage survival sug-
gests that ABH8-catalyzed tRNA modification could be linked
to the DNA damage response; therefore, we tested whether
ABH8 gene expression is modulated upon exposure to bleo-
mycin. Indeed, bleomycin exposure induced ABH8 expression,
with peak induction occurring at 6 to 12 h postdamage (Fig.
7D). Notably, we find that cells depleted of the central up-
stream DNA damage response kinase, ATM, lacked this dy-
namic induction of ABH8 after bleomycin treatment, suggest-
ing that ABH8 induction requires an intact DNA damage
response pathway. The requirement for ABH8 in DNA dam-
age survival and the ATM-dependent modulation of ABH8
expression suggest a link between ABH8, tRNA modification,
and the DNA damage response in human cells.
The correlation between ABH8 depletion and DNA damage
sensitivity suggests that maintenance of proper tRNA modifi-
cations is necessary for DNA damage survival. Thus, to provide
additional evidence that modification of the tRNA wobble
position is required for DNA damage survival, we analyzed the
effect of depleting URM1 (ubiquitin-related modifier 1), a
small sulfur carrier protein that is required for the thiol mod-
ification of tRNAs at the wobble positions, including those of
tRNAGlu(UUC)and tRNAArg(UCU)(29, 40). Strikingly, we find
that URM1 depletion leads to an MMS sensitivity phenotype
FIG. 7. Depletion of ABH8 diminishes cellular survival after exposure to DNA-damaging agents. (A) Colony formation assay of human cell
lines after treatment with the indicated doses of MMS. (B and C) Viability of control and ABH8-depleted human cell lines after treatment with
the indicated doses of MMS or bleomycin as measured by trypan blue dye exclusion or by the WST-1 cell proliferation and viability assay.
(D) Verification of full-length ABH8 expression in human cell lines. Whole-cell extracts prepared from the indicated human cell lines were
immunoblotted with antibody probes against the respective proteins. The asterisk denotes a protein that cross-reacts with the anti-ABH8 antibody.
(E and F) Viability of human cell lines after treatment with MMS or bleomycin. (G) Real-time RT-PCR analysis of ABH8 transcripts from the
indicated human cells. (H) Confirmation of URM1 depletion in human cells. (I) Viability of control and URM1-depleted human cell lines after
treatment with MMS.
VOL. 30, 2010 tRNA METHYLATION BY THE HUMAN AlkB HOMOLOG ABH82457
comparable to that of ABH8-depleted cells (Fig. 7H and I).
These results provide independent evidence that proper mod-
ification of wobble uridines in specific tRNAs is required for
DNA damage survival.
The formation of diverse tRNA modifications requires many
cellular pathways involving numerous enzymes. In mammals,
several putative tRNA methyltransferases have been identified
by sequence homology with bacterial or yeast proteins (11), but
the majority of these enzymes remain uncharacterized, and
their requirement in tRNA methylation awaits verification.
Interestingly, the DNMT2 enzyme of the DNA cytosine meth-
yltransferase family is one of the few known mammalian pro-
teins with a confirmed role in tRNA methylation (20). DNMT2
methylates tRNAAspusing a DNA methyltransferase-like cat-
alytic mechanism, but the function of this modification is un-
known. Here, we show that human ABH8 protein contains an
active RNA methyltransferase domain that is essential for
maintaining the levels of a critical tRNA modification in hu-
In addition to tRNA methylation, the DNA damage sensi-
tivity phenotype of ABH8-depleted cells suggests a role for
ABH8 in DNA damage survival through the regulation of
tRNA modification, potentially in concert with any oxidative
demethylase activity conferred by the AlkB dioxygenase do-
main of ABH8. Support for this notion comes first from our
confirmation that ABH8 localizes primarily to the cytoplasm
and second from our finding that ABH8 protects against bleo-
mycin, a damaging agent that does not methylate nucleic acids.
Future studies will be devoted to understanding the relative
contributions of the ABH8 dioxygenase and methyltransferase
domains to survival after DNA damage.
In yeast, mRNA transcripts encoding stress response pro-
teins are significantly enriched for codons decoded by tRNAs
containing the Trm9p-dependent mcm5U wobble modification
(6). The mcm5U wobble base generated by Trm9 has been
shown to modulate tRNA-mRNA pairing and enhance binding
with the cognate codon (2). Thus, tRNA modification enzymes
can directly regulate the translation of specific proteins by
modulating a subset of codon-anticodon interactions. Our re-
sults indicate that a conserved mechanism in which ABH8-
catalyzed tRNA modification regulates the translation of spe-
cific proteins that are essential for surviving genotoxic stress
could be operating in human cells. Notably, previous reports
have demonstrated the preferential translation of mRNAs en-
coding DNA damage response and repair proteins in human
cells after cellular stress (37). Due to the presence of mcm5U
in the wobble position of tRNASec(UGA), ABH8 could also
modulate the specific translation of the entire repertoire of
selenocysteine proteins. It will be of great interest to identify
the specific proteins whose translation is regulated by tRNA
modification catalyzed by ABH8.
The fusion of a putative dioxygenase motif to an active meth-
yltransferase domain in ABH8 suggests the intriguing possibility
of reversible RNA modification. While the substrates of the
ABH8 dioxygenase motif remain to be discovered, ABH8 could
demethylate wobble uridines or other modified nucleosides in
tRNA under particular conditions. Alternatively, given the RNA
repair capacity demonstrated by certain AlkB proteins (1, 34),
ABH8 could reverse aberrant methylation of tRNA, either
caused by spurious methylase activity of the methyltransferase
domain or induced by endogenous or exogenous alkylating
agents. Alkylating agents have been shown to alter or inacti-
vate mRNA, tRNA, or rRNA function, leading to ribosome
stalling, miscoding, or translational blocks, with the production
of truncated or mutant polypeptides (32, 34). While TRiC
could function as a chaperone for the correct folding of a
multidomain protein such as ABH8, it could also represent a
mechanism by which incompletely synthesized translation
products caused by a stalled ribosome are stabilized by TRiC
while ABH8 rescues the damaged RNAs.
The potential substrates of ABH8 could also encompass
protein targets since the AlkB enzymes belong to a superfamily
of iron-dependent dioxygenases that include the JmjC domain
histone demethylases, which have an enzymatic mechanism
identical to that of AlkB to demethylate lysine residues in
histone proteins (35). In addition, oxidative cleavage of amino
acids by iron-dependent dioxygenases such as cysteine dioxy-
genase plays important roles in maintaining proper levels of
particular amino acids (25). In the case of ABH8, oxidative
cleavage of specific amino acids or proteins could occur if their
levels are upregulated under particular conditions of stress or
Of significance, ABH8 is highly expressed in many urothelial
cancers, with a positive correlation between ABH8 expression
and high-grade, invasive carcinomas (46). Consistent with these
observations, silencing of ABH8 significantly suppresses the
angiogenesis and growth of bladder cancers in vivo (42).
Thus, tRNA modifications catalyzed by methyltransferases
such as ABH8 could be an important factor in the growth and
survival of both normal and transformed cells.
We thank Peter Svensson and Emma Wang for the URM1 knock-
down cell line and the Klungland and Falnes labs for sharing unpub-
This work was supported by NIH grants CA055042 and ES002109 to
L.D.S. and ES01701 to T.J.B., by the Intramural Research Program of
the NIH (R.S.P.), and by the MIT Westaway Fund and NCRR grant
S10-RR023783 to P.C.D. D.F. is supported by an American Cancer
Society TJX Postdoctoral Fellowship, and L.D.S. is an American Can-
cer Society Research Professor.
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